Hematopoietic stem cells (HSCs) regenerate the peripheral blood throughout life via their capacity to both self-renew and differentiate. A critica role for DNA methyltransferases in maintaining effective HSC function has recently come to light through a series of studies in mouse and man. Our laboratory recently found that that DNA methyltransferase 3a (DNMT3A) was highly expressed in murine HSCs, and its conditional loss promoted dramatic self-renewal at the expense of differentiation. In humans, DNMT3A was found to be mutated in around 10% of myelodysplastic syndrome (MDS) patients, and around 20% of patients with either myeloid or lymphoid leukemia. DNMT3A mutations appear to arise in the HSCs, conferring an advantage to mutant cells, as these mutations are found in non-malignant as well as malignant clones, and HSC progeny of all lineages. Finally, cells bearing DNMT3A mutations are common in the peripheral blood of individuals over the age of 50 who exhibit a clonal dominance in their peripheral blood. Together, these studies from both mouse and humans incontrovertibly point to a central role for DNMT3A in maintaining normal hematopoiesis. Nevertheless, how these mutations confer an advantage to HSCs and contribute to clonal expansion as well as to the development of MDS and leukemia remains unclear. Gaining insight into these mechanisms is the overarching goal of this project. Here, we will determine whether DNMT3a mutations lead to blocked differentiation, whether DNA methylation function per se is critical to the hematopoietic impact, and whether different DNMT3A mutations have similar roles. Specifically, we will examine the role in differentiation by mapping DNA methylation changes throughout HSC differentiation, determining the localization of DNMT3A protein throughout the genome, modulating genes involved in self-renewal or differentiation in DNMT3a-KO HSCs, and by modulating DNA methylation. In addition, we will determine whether DNA methylation is required for DNMT3A to exert its role in HSCs. We will add back to KO HSCs specific DNMTs that retain or lack catalysis function, and examining the progeny for DNA methylation and HSC activity. Finally, we will examine commonly occurring DNMT3A mutants such as the R882 form to determine their impact on DNA methylation and hematopoiesis. Together, these studies will definitively link differentiation inhibition with DNMT3A loss-of-function and reveal key targets that could be modulated for therapeutic strategies. These studies will also demonstrate whether catalytic DNA methyltransferase activity is required for full HSC function, and lend insights into the impact of some of common DNMT3A mutations. Our lab is uniquely placed to contribute to this growing area given our pioneering work on the role of DNMT3A in HSCs and our generation of several novel tools and reagents to pursue the proposed questions.